This is a national phase filing of the Application No. PCT/DE97/01333, which was filed with the Patent Corporation Treaty on Jun. 24, 1997, and is entitled to priority of the German Patent Application P 196 25 188.5, filed Jun. 24, 1996.
The present invention relates to a system suitable for the production of AAV vectors, and its use.
For carrying out gene-therapeutic measures it is important to have vectors which can introduce foreign genes into the genomes of cells and which are not toxic for them. An example of such vectors are adeno-associated viruses (AAVs).
AAVs are single-stranded DNA viruses belonging to the parvovirus family. AAVs need helper viruses, particularly adenoviruses or herpesviruses, for their replication. In the absence of helper viruses, AAVs integrate into the host cell genome, particularly at a specific site of chromosome 19.
The genome of AAVs is linear and has a length of about 4680 nucleotides. It comprises two reading frames which code for a structural gene and a non-structural gene. The structural gene is referred to as cap gene. It is controlled by the P40 promoter and codes for three capsid proteins. The non-structural gene is referred to as rep gene and codes for the Rep proteins, Rep 78, Rep 68, Rep 52 and Rep 40. The two former proteins are expressed under the control of the P5 promoter while the expression of Rep 52 and Rep 40 is controlled by the P19 promoter. The functions of the Rep proteins are represented inter alia by the control of the replication and transcription of the AAV genome.
However, the production of AAVs is extremely problematic. In particular, it is difficult to prepare great amounts of recombinant AAVs (rAAVs), i.e., AAVs containing a foreign DNA. Even the attempt of using adenoviruses as vectors for rAAVs does not yield satisfactory results.
Therefore, it is the object of the present invention to provide a product by which rAAVs can be provided in great amounts.
The invention concerns a system comprising an AAV vector, which contains a foreign DNA, and rep 68/78 sequences of AAV with delayed expression, these sequences being present (a) in cis or (b) in trans.
The invention also concerns the use of such a system for the production of AAV vectors.
It is the object of the present invention to provide a product by which rAAVs can be provided in great amounts. According to the invention this is achieved by the subject matters defined in the claims.
Thus, the subject matter of the present invention relates to a system comprising an AAV vector containing a foreign DNA and rep 68/78 sequences of AAV whose expression regarding the DNA replication of the AAV vector or of part thereof is delayed, the rep 68/78 sequences being present (a) in cis or (b) in trans.
The present invention is based on the applicant""s discovery that the Rep proteins 68 and 78 of AAV impair the replication of AAV DNA and that this impairment can be prevented by a delayed expression of the sequences encoding the Rep proteins 68 and 78.
The expression xe2x80x9cAAV vectorxe2x80x9d refers to any AAV vector which contains no rep 68/78 sequences of AAV expressed in non-delayed fashion regarding the DNA replication of the AAV vector or of part thereof. The expression xe2x80x9cpart of the AAV vectorxe2x80x9d relates to any part of the AAV vector, particularly its cap sequences and the sequences coding for the Rep proteins 40 and 52.
The expression xe2x80x9crep 68/78 sequences of AAVxe2x80x9d refers to the fact that the system according to the invention comprises rep 68 sequences and/or rep 78 sequences of AAV whose expression regarding the DNA replication of the AAV vector or of part thereof is delayed.
The expression xe2x80x9cforeign DNAxe2x80x9d comprises any foreign DNA which may be integrated in an above AAV vector. The foreign DNA can be non-coding or coding. In the former case, the foreign DNA may be a regulator or control element of DNA replication and/or transcription. In the latter case, it is favorable for the foreign DNA to be expressible, it being particularly advantageous for the expression to be controlled by a constitutive or inducible promoter such as a tissue-specific or tumor-specific promoter. In addition, the foreign DNA may code for a diagnostic and/or therapeutic protein. Examples of a therapeutic protein are tumor necrosis factor, interferons, interleukins, lymphokines, growth factors, plasma proteins such as coagulation factors and metabolic enzymes, and receptors. In particular, the foreign DNA may code for proteins which can increase the immunogenicity of cells. These may be proteins which are lacking tumor cells, e.g., cytokines, such as IL-2, interferons and GM-CSF, and costimulatory molecules, such as B7-l tumor-associated antigens, e.g., MAGE1, tyrosinases, lymphone-specific idiotypes and viral proteins, e.g., E7 protein of human papilloma virus and EBNA 3 protein of Epstein-Barr virus. The foreign DNA may be integrated at any site of the AAV vector. It may be favorable for several foreign DNAs to be present in one AAV vector.
A system according to the invention comprises in (a) an AAV vector and in cis, i.e., on the AAV vector, the present rep 68/78 sequences of AAV, which are expressed in delayed fashion regarding the DNA replication of the AAV vector or of part thereof. Such an AAV vector can be obtained by common methods. It is favorable to use as a basis an AAV vector which has a DNA coding for Rep proteins of AAV. The endogenic P5 promoter of the rep 68 and rep 78 sequences of AAV can be replaced in such an AAV vector by one which is active after the DNA replication of the AAV vector or of part thereof. Such a promoter is e.g., the xe2x80x9cmajor late promoterxe2x80x9d of adenovirus or a derivative thereof. An inducible promoter, e.g., the metallothioneine promoter or a derivative thereof, can also be used in place of the endogenic P5 promoter of the rep 68 and rep 78 sequences of AAV.
In a preferred embodiment the AAV vector is present in (a) in a product (I), which may be any product that does not impair the functionality of the AAV vector, particularly its replication. Product (I) is favorably a vector, e.g., a virus or plasmid vector, or a cell.
A system according to the invention comprises in (b) an AAV vector and in trans, i.e. separated from the AAV vector, the present rep 68/78 sequences of AAV which are expressed in delayed fashion regarding the DNA replication of the AAV vector or of part thereof. The rep 68/78 sequences of AAV are preferably present in a product (II). It can be any product which does not impair the functionality of the rep 68/78 sequences of AAV, particularly their delayed expression. The latter can be obtained as described in (a) A product (II) is advantageously a vector, e.g., a virus or plasmid vector, or a cell.
It will be particularly favorable if, in (b), the AAV vector is present in a product (I) and the rep 68/78 sequences of AAV are present in a product (II). Products (I) and (II) may have the above meanings, but cannot be cells at the same time. It is advantageous for products (I) and (II) to be virus vectors, e.g., adenovirus vectors or a combination of adenovirus and vaccinia virus vectors. It is particularly favorable for the virus vectors to complement one another. Virus vectors are described below as products (I) and (II). This has to be considered by way of example.
Product (I) is an adenovirus vector which in place of the E1 sequences of adenovirus contains an AAV vector having ITR sequences of AAV and a foreign DNA.
Product (II) is an adenovirus vector which in place of the E3 sequences of adenovirus contains rep and cap sequences of AAV, the rep 68/78 sequences of AAV being controlled by a promoter active after the DNA replication of the AAV vector or of part thereof, e.g., the xe2x80x9cmajor late promoterxe2x80x9d of adenovirus, the rep 40 and rep 52 sequences of AAV being controlled by the endogenic p19 promoter, and the cap sequences of AAV being controlled by a constitutive promoter, e.g., the CMV promoter; product (II) is a combination of two adenovirus vectors, one of which contains rep sequences of AAV in place of the E3 sequences of adenovirus, the rep 68/78 sequences of AAV being controlled by the xe2x80x9cmajor late promoterxe2x80x9d of adenovirus and the rep 40 and rep 52 sequences of AAV being controlled by the endogenic p19 promoter, and the other contains cap sequences of AAV which are controlled by the CMV promoter, in place of the E4 sequences of adenovirus; product (II) is a combination of an adenovirus vector and a vaccinia virus vector, the former containing, in place of the E3 sequences of adenovirus, rep 40 and rep 52 sequences of AAV under the control of the endogenic p19 promoter and cap sequences of AAV under the control of the CMV promoter, and the latter containing rep 68/78 sequences of AAV. The vaccinia virus vector is not added until the DNA replication of the adenovirus vectors is fully or partially complete.
A system according to the invention is suited to prepare recombinant AAVs (rAAVs) in great amounts. For this purpose, the system according to the invention is introduced into cells which permit the replication of rAAVs. These are e.g. 293 cells. The cells are then infected with helper viruses such as adenoviruses. This may be omitted if the system according to the invention provides all elements necessary for the replication of rAAVs. This is found particularly when the system according to the invention is present in alternative (b) in which products (I) and (II) are virus vectors, particularly adenovirus vectors or a combination of adenovirus and vaccinia virus vectors, which complement one another. The complementation may also be given if one of the products is a virus vector and the other product is a cell providing the protein whose DNA is deleted in the virus vector.
By means of the present invention it is possible to provide rAAVs which can be used for diagnostic and/or therapeutic measures. Such measures can be taken in well-calculated fashion by selecting the foreign DNA.
The below examples explain the invention in more detail. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.